Pseudobulk gene expression heatmaps – Schulte-Schrepping et al, 2020, Cell
Pseudobulk gene expression heatmaps – Schulte-Schrepping et al
cohort2
Generate Figure S3F; Figure S4D
input data from pseudobulk eset object – output from pseudobulk object generation pipeline
DGE_IN_PATH <- "../input/SchulteSchrepping/"
g_cohort2.idcelltype <- readRDS(paste(DGE_IN_PATH, "cohort2/cluster_labels_res.0.4.rds", sep = ""))
eset_list <- lapply(g_cohort2.idcelltype, function(dge){
mat <- cpm(dge$counts, log = TRUE)
meta <- dge$samples
ExpressionSet(assayData = mat, phenoData = AnnotatedDataFrame(meta))
})
# get only the first timepoint for each subject
eset_list_T0 <- lapply(eset_list, function(dge){
filter <- colnames(dge)[dge@phenoData@data$Timepoint %in% c("T0", "HC")]
dge$group <- factor(dge$group, levels = c("control","mild","severe"))
dge <- dge[,filter]
})
# read in pathway_summary
pathways <- read.xlsx("../input/selected_pathway_summary.xlsx", sheet = "reduced", startRow = 1)
IFNsets <- filter(pathways, Category %in% c("Type I IFN response",
"Vaccine_infection induced early blood transcriptomic signature"))
Agpresets <- filter(pathways, Primary_gene_sets %in% c("KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION"))
translationsets <- filter(pathways, Category %in% c("Translation/ribosome"))
NFkBsets <- filter(pathways, Primary_gene_sets %in% c("HALLMARK_TNFA_SIGNALING_VIA_NFKB"))
cyclesets <- c("btm_M4.1_cell cycle (I)","btm_M4.0_cell cycle and transcription")
apoptsets <- filter(pathways, Category %in% c("Apoptosis and cell death"))
inflamsets <- filter(pathways, Primary_gene_sets %in% c("HALLMARK_INFLAMMATORY_RESPONSE"))
oxphossets <- filter(pathways, Primary_gene_sets %in% c("KEGG_OXIDATIVE_PHOSPHORYLATION"))
fattysets <- filter(pathways, Primary_gene_sets %in% c("reactome_Fatty acid metabolism"))
chemoksets <- filter(pathways, Primary_gene_sets %in% c("KEGG_CHEMOKINE_SIGNALING_PATHWAY","GO_CELLULAR_RESPONSE_TO_INTERLEUKIN_1"))pDC
apoptosis/cell death
showing the apoptosis signature (union LE genes of COVID19vsHCs and association with DSM) from our cohort that driving the difference of COVID19 and severity
DGE_IN_PATH2 <- "../input/differential_expression/fgsea_tables/"
pDC <- eset_list_T0$pDC
pDC_PC1 <- read.delim(paste0(DGE_IN_PATH2, "PC1/pDC--model@PC1--coef@PC1--fgsea.tsv"), header = TRUE)
pDC_covid <- read.delim(paste0(DGE_IN_PATH2, "COVID-Healthy/pDC--model@healthy_vs_covid--coef@COVID-Healthy--fgsea.tsv"), header = TRUE)
pDC_PC1_apopt <- pDC_PC1 %>% filter(pDC_PC1$pathway %in% apoptsets$Primary_gene_sets)
pDC_covid_apopt <- pDC_covid %>% filter(pDC_covid$pathway %in% apoptsets$Primary_gene_sets)
pDC_PC1_apopt_LE <- unique(unlist(sapply(pDC_PC1_apopt$leadingEdge, function(x) str_split(x, pattern = " "))))
pDC_covid_apopt_LE <- unique(unlist(sapply(pDC_covid_apopt$leadingEdge, function(x) str_split(x, pattern = " "))))
pDC_apopt_LE <- intersect(pDC_PC1_apopt_LE,pDC_covid_apopt_LE)
pDC_apopt_LE_union <- union(pDC_PC1_apopt_LE,pDC_covid_apopt_LE)
pDC_mtx_union <- exprs(pDC)[rownames(exprs(pDC)) %in% pDC_apopt_LE_union,]
meta <- pData(pDC) %>% dplyr::arrange(group,who_per_sample)
pDC_mtx_union <- pDC_mtx_union[,rownames(meta)]
# customize pDC union LE heatmap
# set marked genesets
PC1_COVID <- rownames(pDC_mtx_union) %in% pDC_apopt_LE
oxi_stress_LE <- filter(pDC_PC1, pathway %in% "reactome_Oxidative Stress Induced Senescence")$leadingEdge
oxi_stress <- rownames(pDC_mtx_union) %in% unlist(str_split(oxi_stress_LE, pattern = " "))
names(oxi_stress) <- rownames(pDC_mtx_union)
label <- which(rownames(pDC_mtx_union) %in% union(pDC_PC1_apopt_LE,pDC_covid_apopt_LE)[1:24])
label_genes <- rownames(pDC_mtx_union)[label]
# set colors
col_fun = colorRamp2(c(-1.5, 0, 1.5), c("#FF00FF", "#000000", "#FFFF00"))
who.color <- colorRamp2(c(0, 7), c("white", "#e97171"))
severity.color <- c("control"="#00BA38", "mild"="#619CFF", "severe"="#F8766D")
# set heatmap annotation
ha = HeatmapAnnotation(
severity = meta$group,
who = meta$who_per_sample,
col = list(who = who.color,
severity = severity.color)
)
# pdf("../SchulteSchrepping/output/Subjecthm_pDC_unionLE_apopt_extLE_cohort2.pdf", width = 10, height = 5)
colnames(pDC_mtx_union) <- meta$n_barcodes
p <- Heatmap(pheatmap:::scale_rows(pDC_mtx_union), name = paste("pDC", "apopt", sep = "_"),
show_column_names = TRUE,
top_annotation = ha, cluster_columns = FALSE,
column_split = meta$group,
row_split = PC1_COVID,
col = col_fun,
# row_names_gp = gpar(col = c("blue", "black"), fontsize = c(10, 10)),
column_gap = unit(2, "mm"),
row_gap = unit(2, "mm")
)+
Heatmap(PC1_COVID + 0, name = "coLE", col = c("0" = "white", "1" = "blue"),
show_heatmap_legend = FALSE, width = unit(5, "mm")) +
Heatmap(oxi_stress + 0, name = "OxiSenes", col = c("0" = "white", "1" = "green"),
show_row_names = TRUE, row_names_gp = gpar(fontsize = 8),
show_heatmap_legend = FALSE, width = unit(5, "mm"))+
rowAnnotation(foo = anno_mark(at = label,
labels = label_genes,
labels_gp = gpar(fontsize = 12)))
p
# dev.off()NK cells
Fatty acid metabolism
NK_CD16hi <- eset_list_T0$`NK cells`
# cohort2 LE
NK_CD16hi_PC1 <- read.delim(paste0(DGE_IN_PATH, "cohort2/fgsea_tables/severe-mild/NK cells--model@severity--coef@severe-mild--fgsea.tsv"), header = TRUE)
NK_CD16hi_covid <- read.delim(paste0(DGE_IN_PATH, "cohort2/fgsea_tables/COVID-Healthy/NK cells--model@healthy_vs_covid--coef@COVID-Healthy--fgsea.tsv"), header = TRUE)
NK_CD16hi_PC1_fatty <- NK_CD16hi_PC1 %>% filter(NK_CD16hi_PC1$pathway %in% fattysets$Primary_gene_sets)
NK_CD16hi_covid_fatty <- NK_CD16hi_covid %>% filter(NK_CD16hi_covid$pathway %in% fattysets$Primary_gene_sets)
NK_CD16hi_PC1_fatty_LE <- unique(unlist(sapply(NK_CD16hi_PC1_fatty$leadingEdge, function(x) str_split(x, pattern = " "))))
NK_CD16hi_covid_fatty_LE <- unique(unlist(sapply(NK_CD16hi_covid_fatty$leadingEdge, function(x) str_split(x, pattern = " "))))
NK_CD16hi_fatty_LE <- intersect(NK_CD16hi_PC1_fatty_LE,NK_CD16hi_covid_fatty_LE)
NK_CD16hi_mtx_PC1 <- exprs(NK_CD16hi)[NK_CD16hi_PC1_fatty_LE, ]
meta <- pData(NK_CD16hi) %>% dplyr::arrange(group,who_per_sample)
NK_CD16hi_mtx_PC1 <- NK_CD16hi_mtx_PC1[,rownames(meta)]
# get classifications of FA LE genes
catabolicgene <- read.xlsx("../input/fatty acid LE NK gene categories_cohort2.xlsx", sheet = "catabolic", rowNames = TRUE, colNames = TRUE)$Gene.Symbol
biosyngene <- read.xlsx("../input/fatty acid LE NK gene categories_cohort2.xlsx", sheet = "biosynthetic", rowNames = TRUE, colNames = TRUE)$Gene.Symbol
fatty_NK <- vector(length = nrow(NK_CD16hi_mtx_PC1))
fatty_NK <- replace(fatty_NK, which(rownames(NK_CD16hi_mtx_PC1) %in% biosyngene), "biosynthetic")
fatty_NK <- replace(fatty_NK, which(rownames(NK_CD16hi_mtx_PC1) %in% catabolicgene), "catabolic")
fatty_NK <- replace(fatty_NK, which(fatty_NK == "FALSE"), "others")
names(fatty_NK) <- rownames(NK_CD16hi_mtx_PC1)
# pdf("../SchulteSchrepping/output/Subjecthm_NK_PC1LE_fatty_cohort2.pdf", width = 10, height = 6)
subject.hm.PC1(exprs_mtx = NK_CD16hi_mtx_PC1, meta = meta, celltype = "NK",
module = "fatty", PC1LEset = NK_CD16hi_PC1_fatty_LE,
covidLEset = NK_CD16hi_covid_fatty_LE,
rowsplit=2, othergroup=fatty_NK)
# dev.off()sI <- sessionInfo()
utils:::print.sessionInfo(sI[-c(10,11)])## R version 3.6.1 (2019-07-05)
## Platform: x86_64-conda_cos6-linux-gnu (64-bit)
## Running under: Red Hat Enterprise Linux Server 7.2 (Maipo)
##
## Matrix products: default
##
## locale:
## [1] en_US.UTF-8
##
## attached base packages:
## [1] grid parallel stats graphics grDevices utils datasets
## [8] methods base
##
## other attached packages:
## [1] viridis_0.5.1 viridisLite_0.3.0 circlize_0.4.10
## [4] pheatmap_1.0.12 ComplexHeatmap_2.5.4 openxlsx_4.1.5
## [7] forcats_0.5.0 stringr_1.4.0 dplyr_1.0.0
## [10] purrr_0.3.4 readr_1.3.1 tidyr_0.8.3
## [13] tibble_3.0.3 ggplot2_3.1.1 tidyverse_1.2.1
## [16] Biobase_2.46.0 BiocGenerics_0.32.0 edgeR_3.28.1
## [19] limma_3.42.2
##
## loaded via a namespace (and not attached):
## [1] Rcpp_1.0.5 locfit_1.5-9.4 lubridate_1.7.9
## [4] lattice_0.20-41 png_0.1-7 assertthat_0.2.1
## [7] digest_0.6.25 R6_2.4.1 cellranger_1.1.0
## [10] plyr_1.8.6 backports_1.1.8 stats4_3.6.1
## [13] evaluate_0.14 httr_1.4.2 pillar_1.4.6
## [16] GlobalOptions_0.1.2 rlang_0.4.7 lazyeval_0.2.2
## [19] readxl_1.3.1 rstudioapi_0.11 S4Vectors_0.24.4
## [22] GetoptLong_1.0.2 rmarkdown_2.3 munsell_0.5.0
## [25] broom_0.5.2 compiler_3.6.1 modelr_0.1.4
## [28] xfun_0.15 pkgconfig_2.0.3 shape_1.4.4
## [31] htmltools_0.5.0 tidyselect_1.1.0 gridExtra_2.3
## [34] IRanges_2.20.2 fansi_0.4.1 crayon_1.3.4
## [37] withr_2.2.0 nlme_3.1-148 jsonlite_1.7.0
## [40] gtable_0.3.0 lifecycle_0.2.0 magrittr_1.5
## [43] scales_1.1.1 zip_2.0.4 cli_2.0.2
## [46] stringi_1.4.6 xml2_1.3.2 ellipsis_0.3.1
## [49] generics_0.0.2 vctrs_0.3.2 RColorBrewer_1.1-2
## [52] rjson_0.2.20 tools_3.6.1 glue_1.4.1
## [55] hms_0.5.3 yaml_2.2.1 clue_0.3-57
## [58] colorspace_1.4-1 cluster_2.1.0 rvest_0.3.5
## [61] knitr_1.29 haven_2.1.0